This study examined six gene loci using MLPA-NGS technique. The mutation frequencies were as follows: c.388A > G (93.84%) > c.7G > A (89.23%) > c.597C > T (81.54%) > c.211G > A(69.23%) > c.521T > C(26.15%) > c.175T > C(18.46%). The c.211G > A polymorphism was correlated with severe hyperbilirubinemia, increasing the risk of its development. The results are consistent with previous findings[8–11]. In the present study, the mutation frequency of c.211G > A was 69.23% and allele frequency was 45.38%, which were higher than in Japan (34%) and Vietnam (27.3%)[12]. A study from Vietnam has suggested that total bilirubin levels were higher in c.211G > A homozygous pathogenic variants than in wild type[13]. However, in our study, there was no statistically significant difference between c.211G > A homozygous neonates, heterozygous neonates, and wild-type neonates. This is most likely linked to the fact that this study included only neonates with severe hyperbilirubinemia and not all neonates with hyperbilirubinemia. Unconjugated bilirubin is transported specifically into liver cells using organic anion transporter 2 (OATP2) on liver membrane. OATP2 is encoded by SLCO1B1 gene. Prior research indicated that mutations in this gene cause reduced transport function of the transporter[14]. The c.521T > C has been extensively researched, and the reduced transport function caused by this mutation may be associated with a miscarriage of proteins to the cell membrane[15]. A study from northern China indicated that c.388A > G is linked to severe hyperbilirubinemia occurrence[16]. While Liu J et al. demonstrated that c.388A > G is associated with a higher risk of incident hyperbilirubinemia, there is no significant correlation between C.521T > C and hyperbilirubinemia[17]. Another study revealed that c.388A > G, c.597C > T, and c.521T > C had no link to the incidence of neonatal hyperbilirubinemia in Guangxi[18], consistent with our findings. A study in 2005 discovered that while c.388A > G and G6PD deficiency both contribute to hyperbilirubinemia occurrence, the serum bilirubin concentration is unaffected by c.388A > G mutation only[19]. These findings appear to be contradictory. As a result, a multicenter study with large sample size is warranted. During bilirubin metabolism, hemoglobin is released from human red blood cells and degraded into heme. Heme oxygenase (HO) catabolizes the heme group into CO, biliverdin, and iron; biliverdin is further reduced to bilirubin by BLVRA[20]. Once the genes encoding BLVRA are mutated, the activity of this enzyme is altered, thus affecting bilirubin levels. The results of this study indicate that BLVRA c.7G > A had the highest rate of mutations (89.23%), followed by c.175T > C (18.46%) in the hyperbilirubinemia group, there was no significant correlation between the pathogenic variant and hyperbilirubinemia. There are relatively few studies on this at home and abroad. A 2016 study indicated that c.175T > C were linked to hyperbilirubinemia[21]. Yang et al.'s study on genetic factors of neonatal hyperbilirubinemia revealed that BLVRA gene c.7G > A was not significantly associated with disease's occurrence[22]. Lin et al. investigated the general population of Kazak, Uyghur, and Han Chinese and discovered that C.7G > A mutation was insufficient to alter bilirubin levels[23]. At present, the mechanism of BLVRA gene mutation affecting bilirubin level is incompletely clear and basic experiments must be designed for further study.
In this study, MLPA-NGS technique was employed for the first time to detect genes in neonates with hyperbilirubinemia. The neonates in the hyperbilirubin and control groups were independently sequenced using Sanger sequencing and next-generation sequencing. We compared MLPA-NGS sequencing results with previous sequencing results and discovered that the coincidence rate of UGT1A1c.211G > A was 98.3% (115/117), and all other pathogenic variants had a coincidence rate of 100%. In addition to genetic disease detection, some scholars have recently applied MLPA-NGS technique to high-throughput gene amplification detection of tumor samples, achieving 100% sensitivity and 96% specificity[24, 25]. In this study, we first utilized MLPA-NGS technique to identify neonatal hyperbilirubinemia gene mutation sites. The sequencing results were basically consistent with Sanger sequencing or next-generation sequencing results. An advantage of MLPA-NGS technique is its significantly lower economic cost. It could be promoted as a screening method for mutations at known common loci of neonatal hyperbilirubinemia.
Our study has some limitations. First, the sample size is small. Second, our study was limited to children with severe hyperbilirubinemia, not including children with mild hyperbilirubinemia. I think we can group the children with hyperbilirubinemia according to serum bilirubin levels in future studies to clarify the relationship between bilirubinemia and the above gene mutations.